Structural and functional characterization of monomeric soluble P-selectin and comparison with membrane P-selectin.
Structural and functional characterization of monomeric soluble P-selectin and comparison with membrane P-selectin.
复制标题
DOI：
10.1016/s0021-9258(18)82460-7
复制
发表时间：
1993-07
期刊：
影响因子：
--
通讯作者：
S. Ushiyama;T. Laue;K. Moore;H. Erickson;R. McEver
中科院分区：
文献类型：
--
作者：
S. Ushiyama;T. Laue;K. Moore;H. Erickson;R. McEver
p -选择素是白细胞在凝血酶激活的血小板和内皮细胞上的粘附受体。它包含一个nh2末端碳水化合物识别结构域、一个表皮生长因子基序、9个一致重复序列、一个跨膜结构域和一个细胞质尾部。我们表达了两种可溶形式的p -选择素，一种在第9个重复（tPS）后被截断，另一种由于选择性RNA剪接（asPS）而缺乏跨膜结构域。在电子显微镜下观察，每一个都是单体棒状结构，一端有球状结构域，而来自血小板的膜p -选择素（mPS）形成了球状结构域朝外的莲座。沉降速度和平衡研究证实tPS和asPS是不对称单体，而mPS是寡聚体。HL-60细胞粘附在固定的tPS和asPS上，但粘附效率低于粘附在mPS上。125i标记的tPS和asPS结合约25,000个位点/中性粒细胞和约36,000个位点/HL-60细胞，表观Kd为70 nM。用o -唾液糖蛋白酶处理HL-60细胞可消除asPS的结合位点。我们得出结论：1)p -选择素是一种刚性的、不对称的蛋白质；2)单个可溶性p选择素与白细胞上唾液化的o -低聚糖高亲和力配体结合；3) mPS的寡聚化增强了其对白细胞的亲和力。
P-selectin is an adhesion receptor for leukocytes on thrombin-activated platelets and endothelial cells. It contains a NH2-terminal carbohydrate-recognition domain, an epidermal growth factor motif, nine consensus repeats, a transmembrane domain, and a cytoplasmic tail. We expressed two soluble forms of P-selectin, one truncated after the ninth repeat (tPS) and the other lacking the transmembrane domain due to alternative RNA splicing (asPS). When visualized by electron microscopy, each was a monomeric rod-like structure with a globular domain at one end, whereas membrane P-selectin (mPS) from platelets formed rosettes with the globular domains facing outward. Sedimentation velocity and equilibrium studies confirmed that tPS and asPS were asymmetric monomers, whereas mPS was oligomeric. HL-60 cells adhered to immobilized tPS and asPS, although less efficiently than to mPS. 125I-Labeled tPS and asPS bound to approximately 25,000 sites/neutrophil and approximately 36,000 sites/HL-60 cell with an apparent Kd of 70 nM. Treatment of HL-60 cells with O-sialoglycoprotease eliminated the binding sites for asPS. We conclude that 1) P-selectin is a rigid, asymmetric protein; 2) monomeric soluble P-selectin binds to high affinity ligands with sialylated O-linked oligosaccharides on leukocytes; and 3) oligomerization of mPS enhances its avidity for leukocytes.